A key area of my research is focused on the design and development of optical methodology including advanced light microscopy techniques to investigate/monitor exogenous and endogenous protein-protein interactions, in vivo/intravital imaging and monitoring the physical parameters of normal versus cancer tissues. I am the founder and center director of the W.M. Keck Center for Cellular Imaging (KCCI), an internationally known center in advanced optical and microscopy imaging, particularly in the area of protein-protein interactions in living and fixed specimens. We developed a 2- and 3-color steady state, and FLIM based Förster resonance energy transfer (FRET) imaging system for protein localization in living specimens. We developed a PFRET software package for quantitative FRET data analysis to estimate the nanometer (1-10 nm) distance between the protein molecules in living/fixed cells and tissue for various light microscopy techniques for any combination of fluorophore pairs. Recently, we developed FLIRR method to estimate the metabolic redox ratio in tissue, animal and cells. I am one of the pioneers in developing lifetime imaging microscopy for intracellular calcium measurement and later I developed the same methodology for protein-protein interactions and cancer diagnosis. I have published over 135 articles in refereed journals and book chapters. I have given more than 150 invited lectures nationally and internationally. I have edited three books, Chairperson in organizing an annual International conference on Multiphoton Microscopy in the Biomedical Sciences through SPIE (since 2001) and runs a hands on training annual workshop on FRET Microscopy at the University of Virginia, Charlottesville during March (since 2002). I was elected “Fellow” members of the SPIE.
This will count as one of your downloads.
You will have access to both the presentation and article (if available).
JBO guest editors introduce the Special Section Celebrating Thirty Years of Multiphoton Microscopy in the Biomedical Sciences.
Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)—essential coenzymes in cellular respiration—have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.
This will count as one of your downloads.
You will have access to both the presentation and article (if available).
This course introduces the basics of multiphoton microscopy, technology development, image acquisition and analysis related to various biological and clinical applications. The first part of the course will cover the basics of microscopy, image formation, detectors, lenses used in MM, and calculation of absorption cross-sections. The second part of the course will focus on how to build your own MM, or integrate one with an existing confocal microscope. The tricks involved in MM development and imaging will be covered. Finally, we will review many applications including tissue imaging, MM in Neuro and developmental biology, labeling issues for MM imaging, FLIM, FRET and other clinical applications.
View contact details
No SPIE Account? Create one
